Technical Field
The present invention relates generally to the field of sophorolipids (SL) and more specifically to new compositions of matter for uses of modified sophorolipids (MSL) and combination of sophorolipids as solubilizing agents, emulsifiers, dispersants and thereof.
Prior Art
Sophorolipids (SL) are glycolipid biosurfactant molecules produced by yeasts, such as Candida bombicola, Yarrowi alipolytica, Candida apicola, and Candida bogoriensis. Microbial biosurfactants are surface active compounds produced by various microorganisms. They lower surface and interfacial tension and form spherical micelles at and above their critical micelle concentration (CMC). Microbial biosurfactants generally have an amphiphilic structure, possessing a hydrophilic moiety, such as an amino acid, peptide, sugar or oligosaccharide, and a hydrophobic moiety including saturated or unsaturated lipid or fatty acids.
SLs consist of a hydrophilic carbohydrate head, sophorose, and a hydrophobic fatty acid tail with generally 16 or 18 carbon atoms with saturation or unsaturation. Sophorose is an unusual disaccharide that consists of two glucose molecules linked β-1,2. Furthermore, sophorose in SLs can be acetylated on the 6′- and/or 6″-positions (FIG. 1). One fatty acid hydroxylated at the terminal or subterminal (β-1) positions is β-glycosidically linked to the sophorose molecule. The fatty acid carboxylic acid group is either free (acidic or open form) or internally esterified generally at the 4″-position (lactonic form) (FIG. 1). The hydroxy fatty acid component of SLs generally has 16 or 18 carbon atoms with generally one unsaturated bond (Asmer et al. 1988; Davila et al. 1993). However, the SL hydroxy fatty acid can also be fully saturated, di-unsaturated or tri-unsaturated. As such, SLs synthesized by C. bombicola consist of a mixture of related molecules. Differences between these molecules are found based on: i) the fatty acid structure (degree of unsaturation, chain length, and position of hydroxylation), whether they are produced in the lactonic or ring-opened form, and ii) the acetylation pattern.
Studies have been carried out to “tailor” SL structure during in vivo formation. These studies have mainly involved the selective feeding of different lipophilic substrates. For example, changing the co-substrate from sunflower to canola oil resulted in a large increase (50% to 73%) in the lactonic portion of SLs (Tulloch et al. 1962; Asmer et al. 1988; Davila et al. 1992; Zhou et al. 1992, 1995). Also, unsaturated C-18 fatty acids of oleic acid may be transferred unchanged into SLs (Rau et al. 1999). Finally, lactonic and acidic SLs are synthesized in vivo from stearic acid with similar yields to oleic acid-derived SLs (Felse et al. 2007). Thus, to date, physiological variables during fermentations have provided routes to the variation of SL compositions.
As noted above, fermentation by different microorganisms, Candida bombicola, Yarrowi alipolytica, Candida apicola, and Candida bogoriensis, leads to sophorolipids of different structure noted above, the variations in sophorolipids based on fatty acid feedstocks and organisms leads to a wide array of sophorolipids including lactonic and acidic structures. An additional modification that is relevant to acidic sophorolipids is cleavage of the sophorose moiety to the corresponding glucose-based glucolipids. Treatment of acidic sophorolipids with enzymes β-glucuronidase (Helix pomatia), cellulase (Penicillium funiculosum), Clara diastase (a mixture of enzymes including amylase, cellulase, peptidase, phosphatase, and sulphatase), galactomannanase (Aspergillus niger), hemicellulase (Aspergillus niger), hesperidinase (Aspergillus niger), inulinase (Aspergillus niger), pectolyase (Aspergillus japonicus), or naringinase (Penicillium decumbens) afford glucolipids over a range of pH values (Rau et al. 1999) (for enzymatic treatment of SLs see FIG. Scheme 1).
In addition to tailoring SL in vivo formation, it is known that by chemical or enzymatic modification of SLs, their physical properties can be manipulated (Zhang et al., 2004). Modifications of SLs were performed so that the chain length of the n-alkyl group (methyl, ethyl, propyl, butyl, and hexyl) esterified to the SL fatty acid was varied. The effect of the n-alkyl ester chain length on interfacial properties of corresponding sophorolipid analogues was studied. The critical micelle concentration (CMC) and minimum surface tension have an inverse relationship with the alkyl ester chain length. That is, CMC decreased to ½ per additional CH2 group for the methyl, ethyl, and propyl series of chain lengths. These results were confirmed by fluorescence spectroscopy. Adsorption of SL alkyl esters on hydrophilic solids was also studied to explore the type of lateral associations. These surfactants were found to absorb on alumina but much less on silica. This adsorption behavior on hydrophilic solids is similar to that of sugar-based nonionic surfactants and unlike that of nonionic ethoxylated surfactants. Hydrogen bonding is proposed to be the primary driving force for adsorption of the sophorolipids on alumina. Increase in the n-alkyl ester chain length of SLs caused a shift of the adsorption isotherms to lower concentrations. The magnitude of the shift corresponds to the change in CMC of these surfactants.
It has been shown that modified sophorolipids (MSLs) have antibacterial, antiviral, and anti-inflammatory properties (Mueller et al. 2006; Shah et al. 2005). In one example, MSLs were shown to down-regulate expression of pro-inflammatory cytokines including interleukin (Hagler et al. 2007). Furthermore, as shown in Table 1, the antibacterial activity of SLs can be increased by up to 1,000 times relative to the natural SL mixture by simple modifications such as esterification of fatty acid carboxyl groups and selective acetylation of disaccharide hydroxyl groups. Table 1 comprises a table of sophorolipid derivatives and sophorolipid components of the natural mixture used in bacterial and fungal plant pathogen assays. The hydroxylated fatty acid of the natural mixture is predominantly 17-hydroxyoleic acid. However, other fatty acid constituents with variations in chain length and unsaturation may also be present.
Our previous work on antimicrobial activity of MSLs showed antimicrobial activity against plant pathogens that include fungi, bacteria and their spores at 0.15 to 10 mg/mL minimum inhibitory concentrations (MIC) (U.S. patent application Ser. No. 12/360,486 and U.S. Provisional Patent Application No. 61/320,885). Further, formulation of MSLs with TWEEN® 20 brand of surface active agent and Polypropylene glycol increased the broad spectrum antimicrobial activity of SLs (U.S. Provisional Patent Application No. 61/543,122). While preparing MSL for formulation we noticed a surprising result, i.e., when we mix two or more modified SLs (e.g., compound 6 with 7), or modified sophorolipids with a natural sophorolipids, there is an increase in the solubility for the combined compounds in distilled water relative to these compounds individually. For example, a 1:1 mol/mol mixture of compound 6 and 7 in distilled water without any additives (e.g. TWEEN® 20 and Polypropylene glycol) was soluble up to 10 mg/L whereas the solubility of compounds 6 and 7 is less than 1 mg/L when they were studied separately/individually. Furthermore, when mixing modified SLs (e.g., compound 6 with 7), or modified sophorolipids with natural sophorolipids, there were extraordinary increases in mixture antimicrobial activity (U.S. patent application Ser. No. 13/757,762) relative to the antimicrobial activity for each of the components when presented individually. Moreover, we discovered that mixing modified SLs or a modified sophorolipids with a natural sophorolipids can result in enhanced performance properties for other applications. Furthermore, we also discovered that unexpected enhanced performance in specific applications may be discovered by exploring the properties of a modified sophorolipid library.
US Patent Publication. No. 2010/0098821 A1 discloses a process that solubilizes essential oils to produce clear beverages using ionic and non-ionic emulsifiers. The process described in this invention simplifies the introduction of normally insoluble nutraceuticals, particularly lipophilic ones, into beverages. Many ionic and non-ionic surfactants were described for use in formation of these emulsions such as sorbitan esters, polyglycerol esters, monoglyceride esters, diglyceride esters, polyethylene glycol esters, sucrose esters, dioctyl sodium sulfosuccinate and lecithin. Neither modified nor natural sophorolipids are mentioned in US Patent Publication. No. 2010/0098821 A1. Furthermore, because the composition of a flavor oil depends on its origin and processing, the most effective compounds for its solubilization cannot be anticipated by one skilled in the art. Hence, the availability of safe and effective compounds that prove useful for solubilization of various flavor and fragrance oil compositions are highly useful to formulators. This present disclosure provides methods for the formulation of micro- and nanoemulsions using one or mixtures of modified sophorolipids, mixtures of modified sophorolipids and natural sophorolipids, as well as mixtures containing one or move modified and/or natural sophorolipid with other molecules that are already known by one skilled in the art as being useful for solubilization of flavor and fragrance oils.
U.S. Pat. No. 6,214,957 B1 disclosed the use of solubilizers, emulsifiers and dispersing agents having the effects of moisturizing the skin when used as washing agents and having the character of elevating the concentration of a material to be solubilized, emulsified or dispersed in solvents including water as compared with the case where the material is employed alone, or elevating the apparent concentration of a material to be emulsified or dispersed in solvents by increasing homogeneity of the emulsion or dispersion which contain as the active ingredient polymers obtained by polymerizing monomer compositions containing at least one hydrophilic monomer (a i.e., 2-(methacryloyloxy)ethyl-2′-(alkyl-substituted or non-substituted ammonio)ethyl phosphate) having a group represented by general formula (1) in the side chain, wherein R1, R2 and R3 represent each H or C 1-4 alkyl group having 1 to 4 carbon atoms, and are the same or different groups) and, as a hydrophobic monomer (b), 0 to 80 weight % of (meth)acrylate. Furthermore, the solubilizers, emulsifiers and dispersing agents having the effects of moisturizing the skin described in U.S. Pat. No. 6,214,957 B1 have no or low bio-based content and are not fully biodegradable. In contrast, the present disclosure discloses MSLs with different chemical compositions that can be used in various combinations of other compounds, are highly effective molecules for a wide range of oil types such as fragrance, flavor and nutraceuticals compounds. Furthermore, modified sophorolipids are fully or highly bio-based and are completely biodegradable when disposed in many environments including in waste-water systems.
The present inventions suggest that by careful modulation of the SL structure via simple chemical modification methods, dramatic shifts in their interfacial activity can be achieved that allow them to be “tuned” or optimized so that highly effective MSL compounds are obtained for applications as solubilizing agents, emulsifiers, dispersants and thereof. The inventors are aware of no examples in prior patents or other literature sources that describe the method developed herein that comprises the development of a library of modified sophorolipids using a wide-range of chemical and enzyme catalyst tools to identify MSLs that can be used in pure form, as mixtures with other modified sophorolipids, as mixtures with natural sophorolipids, as mixtures with modified and natural sophorolipids, and as mixtures with other compounds known by one skilled in the art for use in the dispersion, solubilization or emulsification of various oil types and nutraceuticals.
The new solubilizing, emulsification and dispersant activities of MSLs and various combinations described above that are disclosed herein are unique in structure relative to known sophorolipid derivatives in previous art. It is to these needs and others that the present invention is directed.